Optical imaging techniques such as fluorescence and bioluminescence imaging which use light source at different wavelengths for image generation, provide a simple and direct visualization of specific molecular targets or biological pathways in vitro and in vivo. Fluorescent technique requires the use of small molecule reporters such as fluorogenic probes which provide a measurable optical signal for a particular enzyme facilitated molecular process. Fluorescent detection is more advantageous in compared to colorimetric or radioisotope assay due to its high sensitivity, relative safety, low cost and easy handling. Therefore, imaging of metabolic and signaling events in live cells represent an important frontier in this field of fluoroprobes.
Certain probes can consist of fluorogenic or fluorescent dyes coupled to a blocking group thus forming an enzyme substrate. These probes are cleavable by enzymes, and produce fluorescent dye precipitates, which may be detected by fluorescence microscopy. For example, fluorogenic substrates can be detected by hydrolases which may be used in enzyme labeled fluorescence assays, where the fluorescence is used to detect enzyme activity. Examples of known fluorogenic substrates include 4-methylumbelliferyl β-D-galactoside for detecting β-D-galactosidase (GAL) enzyme activity and 4-methylumbelliferyl β-D-glucuronide for detecting β-D-glucuronidase (GUS) enzyme activity (Ishikawa E. et al, J. Biochem 73, 1319-1321, 1973; Jefferson R. A. Nature, 342, 837-838, 1989). Other fluorogenic substrates that are made from a class of fluorophores generally including quinazolinonoes for example, can be enzymatically converted to a detectable phenolic product, e.g. formation of a soluble coloured or fluorescent product or formation of a precipitate. For example, U.S. Pat. No. 5,316,906 describes such a substrate which consists of substances coupled with phosphate, sulfate or sugar groups and which form a highly fluorescent precipitate upon reaction with an appropriate enzyme. Particular examples of these substrates include ELF97® β-D-galactosidase substrate (ELF97® β-D-galacto-pyranoside) and ELF97® β-D-glucuronide that are commercially available.
However, the potential disadvantages of known fluorogenic substrates are their limited permeability for cell membranes and higher background caused by the autofluorescence of cell and tissue and unstable substrates. As a result, GUS and GAL assays are generally destructive for cells since the cells need to be permeabilized prior to detection of an intracellular analyte, and are thereof not suitable under in vivo conditions, or under in vitro conditions where cell integrity is desired. Many other substrates are not photostable, such as fluorescein, which bleaches after a few minutes, losing the fluorescence necessary for detection.
Monoamine oxidases (MAOs) are essential FAD-dependent enzymes in the living systems and play an essential role in the regulation of monoamine neurotransmitters such as dopamine and serotonin. It can efficiently catalyze the oxidation deamination of neuro transmitters and biogenic amines, to the corresponding imines which are released from the enzyme and hydrolyzed to the corresponding aldehydes. There are two isoforms, MAO A and MAO B, found large in abundant in the liver, gastrointestinal tract, blood platelets and central nervous systems. These enzymes play an important role in metabolism and neural development by regulating the homeostasis of amine neurotransmitters and periphery dietary amines. Any excess or deficiency of these enzyme activities will lead to various neurological and psychiatric disorders such as depression, Parkinson, Alzheimers's diseases or even the growth inhibition and progression of cancer. As such, development of suitable MAOs assays which enable selectively and sensitively monitoring of enzyme activities in complex biological system is of a highly fundamental necessity.
Despite its importance, currently available methods to monitor monoamine oxidase activity involving the use of colorimetric, radioisotope, require a secondary activating enzyme to release the signal for the detection or do not provide single-cell enzyme imaging. A fluorescence probe based on a coumarin derivative for detecting monoamine oxidase activity has been developed (see US Publication 2008/0194522), However, the possible photodamage, relative higher autofluorescence from most cell and tissue would be the potential issues for the further living cell studies. Although several methods based on fluorescent and bioluminescent detection have been developed in monitoring MAO activities, till date no report has been made in providing a direct and sensitive, real time imaging of MAO in living cells systems.
Therefore, it is an object of the present invention to provide an alternative fluorogenic probe which can efficiently detect enzyme activity in living cell systems.
The object is solved by a compound of general formula (I) as defined in the appended claims.